Calcium, the fifth most abundant element in the human body, plays an important physiological role and is essential for both the functional integrity of the nervous and muscular systems as well as normal cardiac function. Calcium is also one of the factors that operates in the mechanisms involved in the coagulation of the blood. More than 90% of the calcium in the human body is in the skeleton as calcium phosphate and calcium carbonate--the remainder of calcium is present in plasma. Plasma calcium levels vary from between about 9.0 and about 11.0 mg/100 mL. On average, about 46% is bound to protein; slightly more is free and ionized; and the remainder is freely diffusible but complexed and ionized. Total calcium, or "CAT" is the combined value of bound calcium ("CAB") free calcium ("CaF" or "Ca.sup.++ ") and protein complexed calcium ("CAP"), or: EQU CaT=CaB+CaF+CaP
CaF is generally accepted as being the physiologically active form of calcium in sera. Potentiometric sensors in the form of ion selective electrodes are useful in the determination of CaF.
Ion selective electrode technology involves the use of a reference electrode and an ion selective electrode ("ISE") separated by a membrane which are simultaneously immersed in a sample of a solution containing the desired ion. This simultaneous immersion leads to a potential across the membrane between the electrodes, which potential is proportional to the presence of the desired ion. Most often the investigator desires to only measure the concentration of one ion out of many different ions in solution. Thus, the composition of the ion selective electrode ISE must be capable of transporting the desired ion across the membrane in preference to all other ions which may be present. Two methodologies are associated with ISE technology: direct, where the sample is analyzed directly; and indirect, where the sample is diluted prior to analysis.
Conventional calcium ISE's only measure the free form of calcium. Accordingly, calcium indirect ISE methodologies employ diluents to displace the protein bound to calcium in an attempt to obtain an accurate reading of free calcium. Such conventional methodologies typically make use of strongly acidic diluents prior to ISE measurement in order to denature proteins bound to the calcium, that is, to displace or, "release", the bound calcium.sup.(1). However, some ions, such as citrate (normally present in human sera at a level of about 0.1 mMol/L but during blood transfusions can be present in levels up to about 5 mMol/L), remain effective in binding calcium even under such acidic conditions.sup.(2). Additionally, certain calcium ionophores will not function properly under such acidic conditions. Other methodologies have been described for releasing bound calcium.sup.(3), (4), (5), (6). All of the preceding references of this paragraph are incorporated herein by reference.
Because of the problems associated with denaturing proteins bound to calcium in clinical samples, a diluent that does not rely upon denaturation and that does not utilize conditions which have an adverse affect upon the calcium ISE, as well as analytical methodologies employing such diluents, are desirable and would be of great value in the determination of total calcium in a clinical sample.